Protein kinases constitute a large family of structurally related enzymes that are responsible for the control of a wide variety of signal transduction processes within the cell (Hardie and Hanks (1995) The Protein Kinase Facts Book. I and II, Academic Press, San Diego, Calif.). The kinases may be categorized into families by the substrates they phosphorylate (e.g., protein-tyrosine, protein-serine/threonine, lipids, etc.). Sequence motifs have been identified that generally correspond to each of these kinase families (e.g., Hanks and Hunter, FASEB J., (1995) 9. 576-596; Knighton, et al., Science, (1991) 253, 407-414; Hiles, et al., Cell, (1992) 70, 419-429; Kunz, et al., Cell, (1993) 73, 585-596; Garcia-Bustos, et al., EMBO J., (1994) 13, 2352-2361).
Protein kinases may be characterized by their regulation mechanisms. These mechanisms include, for example, autophosphorylation, transphosphorylation by other kinases, protein-protein interactions, protein-lipid interactions, and protein-polynucleotide interactions. An individual protein kinase may be regulated by more than one mechanism.
Kinases regulate many different cell processes including, but not limited to, proliferation, differentiation, apoptosis, motility, transcription, translation and other signalling processes, by adding phosphate groups to target proteins. These phosphorylation events act as molecular on/off switches that can modulate or regulate the target protein biological function. Phosphorylation of target proteins occurs in response to a variety of extracellular signals (hormones, neurotransmitters, growth and differentiation factors, etc.), cell cycle events, environmental or nutritional stresses, etc. The appropriate protein kinase functions in signalling pathways to activate or inactivate (either directly or indirectly), for example, a metabolic enzyme, regulatory protein, receptor, cytoskeletal protein, ion channel or pump, or transcription factor. Uncontrolled signalling due to defective control of protein phosphorylation has been implicated in a number of diseases, including, for example, inflammation, cancer, allergy/asthma, disease and conditions of the immune system, disease and conditions of the central nervous system, and angiogenesis.
Aurora Kinases
Three members of the Aurora kinase family have been found in mammals so far (Nigg, Nat. Rev. Mol. Cell Biol. (2001) 2, 21-32). Aurora A kinase (also referred to in the literature as Aurora 2) is a serine/threonine kinase that is involved in the G2 and M phases of the cell cycle, and is an important regulator of mitosis. Aurora kinase A is believed to play a part in mitotic checkpoint control, chromosome dynamics and cytokinesis (Adams et al., Trends Cell Biol., (2001) 11, 49-54). The kinases are located at the centrosomes of interphase cells, at the poles of the bipolar spindle and in the mid-body of the mitotic apparatus.
The other two currently known Aurora kinases are Aurora B (also referred to in the literature as Aurora 1) and Aurora C (also referred to in the literature as Aurora 3). The Aurora kinases have highly homologous catalytic domains but differ considerably in their N-terminal portions (Katayama et al, Cancer Metastasis Rev. (2003) 22(4), 451-64).
The substrates of the Aurora kinases A and B have been identified as including a kinesin-like motor protein, spindle apparatus proteins, histone H3 protein, kinetochore protein and the tumour suppressor protein p53.
Aurora A kinases are believed to be involved in spindle formation and become localised on the centrosome during the early G2 phase where they phosphorylate spindle-associated proteins (Prigent et al., Cell (2003) 114, 531-535). Hirota et al, (Cell, (2003) 114, 585-598) found that cells depleted of Aurora A protein kinase were unable to enter mitosis. Furthermore, it has been found (Adams, 2001) that mutation or disruption of the Aurora A gene in various species leads to mitotic abnormalities, including centrosome separation and maturation defects, spindle aberrations and chromosome segregation defects.
Aurora kinase A is generally expressed at a low level in the majority of normal tissues, the exceptions being tissues with a high proportion of dividing cells such as the thymus and testis. However, elevated levels of Aurora kinases have been found in many human cancers (Giet et al., J. Cell. Sci. (1999) 112, 3591 and Katayama (2003)). Furthermore, Aurora A kinase maps to the chromosome 20q13 region that has frequently been found to be amplified in many human cancers.
Thus, for example, significant Aurora A over-expression has been detected in human breast, ovarian and pancreatic cancers (see Zhou et al., Nat. Genet. (1998) 20, 189-193; Tanaka et al., Cancer Res. (1999) 59, 2041-2044 and Han et al., Cancer Res. (2002) 62, 2890-2896).
Moreover, Isola (American Journal of Pathology (1995) 147, 905-911) has reported that amplification of the Aurora A locus (20q13) correlates with poor prognosis for patients with node-negative breast cancer.
Amplification and/or over-expression of Aurora-A is observed in human bladder cancers and amplification of Aurora-A is associated with aneuploidy and aggressive clinical behaviour (see Sen et al., J. Natl. Cancer Inst. (2002) 94, 1320-1329).
Elevated expression of Aurora-A has been detected in over 50% of colorectal cancers (see Bischoff et al., EMBO J. (1998) 17, 3052-3065 and Takahashi et al., Jpn. J. Cancer Res. (2000) 91, 1007-1014), ovarian cancers (see Gritsko et al., Clin. Cancer Res. (2003) 9, 1420-1426) and gastric tumours (see Sakakura et al., British Journal of Cancer (2001) 84, 824-831).
Tanaka et al., (Cancer Research (1999) 59, 2041-2044) found evidence of over-expression of Aurora A in 94% of invasive duct adenocarcinomas of the breast.
High levels of Aurora A kinase have also been found in renal, cervical, neuroblastoma, melanoma, lymphoma, pancreatic and prostate tumour cell lines (Bischoff et al., (1998), EMBO J. (1998) 17, 3052-3065; Kimura et al., J. Biol. Chem. (1999) 274, 7334-7340; Zhou et al., Nature Genetics, 20: 189-193 (1998); Li et al., Clin Cancer Res. 9 (3): 991-7 (2003).
Royce et al (Cancer. (2004) 100(1), 12-19) report that the expression of the Aurora 2 gene (known as STK15 or BTAK) has been noted in approximately one-fourth of primary breast tumours.
Reichardt et al (Oncol Rep. (2003) 10(5), 1275-9) have reported that quantitative DNA analysis by PCR to search for Aurora amplification in gliomas revealed that 5 out of 16 tumours (31%) of different WHO grade (1× grade II, 1× grade III, 3× grade IV) showed DNA amplification of the Aurora 2 gene. It was hypothesized that amplification of the Aurora 2 gene may be a non-random genetic alteration in human gliomas playing a role in the genetic pathways of tumourigenesis.
Results by Hamada et al (Br. J. Haematol. (2003) 121(3), 439-47) also suggest that Aurora 2 is an effective candidate to indicate not only disease activity but also tumourigenesis of non-Hodgkin's lymphoma. Retardation of tumour cell growth resulting from the restriction of this gene's functions could be a therapeutic approach for non-Hodgkin's lymphoma.
In a study by Gritsko et al (Clin Cancer Res. (2003) 9(4), 1420-6), the kinase activity and protein levels of Aurora A were examined in 92 patients with primary ovarian tumours. In vitro kinase analyses revealed elevated Aurora A kinase activity in 44 cases (48%). Increased Aurora A protein levels were detected in 52 (57%) specimens. High protein levels of Aurora A correlated well with elevated kinase activity.
Results obtained by Li et al (Clin. Cancer Res. 2003 March; 9(3):991-7) showed that the Aurora A gene is overexpressed in pancreatic tumours and carcinoma cell lines and suggest that overexpression of Aurora A may play a role in pancreatic carcinogenesis.
Similarly, it has been shown that Aurora A gene amplification and associated increased expression of the mitotic kinase it encodes are associated with aneuploidy and aggressive clinical behaviour in human bladder cancer. (J. Natl. Cancer Inst. (2002) 94(17), 1320-9).
Investigation by several groups (Dutertre and Prigent, Mol. Interv. (2003) 3(3), 127-30 and Anand et al., Cancer Cell. (2003) 3(1), 51-62) suggests that overexpression of Aurora kinase activity is associated with resistance to some current cancer therapies. For example overexpression of Aurora A in mouse embryo fibroblasts can reduce the sensitivity of these cells to the cytotoxic effects of taxane derivatives. Therefore Aurora kinase inhibitors may find particular use in patients who have developed resistance to existing therapies.
On the basis of work carried out to date, it is envisaged that inhibition of Aurora A kinase will prove an effective means of arresting tumour development.
It has also been shown that there is an increase in expression of Aurora B in tumour cells compared to normal cells (Adams et al., Chromasoma. (2001) 110, 65-74). One report suggests that overexpression of Aurora B induces aneuploidy through increased phosphorylation of histone H3 at serine 10, and that cells overexpressing Aurora B form more aggressive tumours and have a higher tendency to form metastatic tumours (Ota et al., Cancer Res. (2002) 62, 5168-5177).
Aurora B is required for both spindle checkpoint function and metaphase chromosome alignment in human cells (Adams et al. J. Cell Biol. (2001) 153, 865-880; Kallio et al., Curr. Biol. (2002) 12, 900-905 and Murata-Hori and Wang Curr. Biol. (2002) 12, 894-899). It has been demonstrated that suppression of Aurora B kinase activity compromises chromosome alignment, spindle checkpoint function and cytokinesis (Ditchfield et al., J. Cell Biol. (2003) 161, 267-280 and Hauf et al., J. Cell Biol. (2003), 161, 281-294). Consequently, after a brief delay cells exit mitosis without dividing and with a 4N DNA content, whereupon they rapidly lose their proliferative potential.
Harrington et al (Nat Med. (2004) 10(3), 262-7) have demonstrated that an inhibitor of the Aurora kinases suppresses tumour growth and induces tumour regression in vivo. In the study, the Aurora kinase inhibitor blocked cancer cell proliferation, and also triggered cell death in a range of cancer cell lines including leukaemic, colorectal and breast cell lines. In addition, it has shown potential for the treatment of leukemia by inducing apoptosis in leukemia cells. VX-680 potently killed treatment-refractory primary Acute Myelogenous Leukemia (AML) cells from patients (Andrews, Oncogene (2005) 24, 5005-5015).
Manfredi et al (PNAS (2007) 104, 4106-4111) have demonstrated that a small-molecule inhibitor of Aurora A suppresses tumour growth in vivo. In the study, dose-dependent tumour growth inhibition was demonstrated in HCT-116 tumour bearing mice and PC-3 tumour bearing mice versus vehicle treated mice. Tumour growth inhibition of up to 84% against HCT-116 and 93% against PC-3 cell xenografts was observed.
Mortlock et al (Clin Cancer Res. (2007) 13(12), 3682-3688) have demonstrated that a small molecule inhibitor of Aurora B suppresses tumour growth in vivo. Immunodeficient mice bearing established SW620, HCT-116, Colo205, A549, Calu-6 or HL-60 tumour xenografts were dosed over 48 h via sub-cutaneous mini-pump infusion with the small molecule inhibitor AZD1152. The inhibition of tumour growth in all cases ranged from 55% to 100% with complete tumour regression observed in 8 of 11 animals bearing the HL-60 xenograft.
On the basis of evidence obtained to date, it is considered likely that Aurora kinase inhibitors should be particularly useful in arresting tumour development and treating cancers such as breast, bladder, colorectal, pancreatic and ovarian cancers, non-Hodgkin's lymphoma, gliomas, nonendometrioid endometrial carcinomas, Acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukaemia (CML), B-cell lymphoma (Mantle cell), and Acute Lymphoblastic Leukemia (ALL).
FLT3
FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase involved in the proliferation, differentiation and apoptosis of hematopoietic and non-hematopoietic cells (Scheijen and Griffin, Oncogene (2002) 21, 3314-3333 and Reilly, British Journal of Haematology (2002) 116, 744-757). As a result of the natural ligand (FL) binding, the FLT3 receptor dimerises resulting in activation of its tyrosine kinase domain, receptor autophosphorylation and recruitment of downstream signalling molecules such as the p85 subunit of PI3K (phosphatidylinositol 3 kinase), PLC-gamma (Phospholipase-C gamma), STAT5a (signal transducer and activator of transcription 5a), and SRC family tyrosine kinases (Gilliland and Griffin, Blood (2002) 100(5), 1532-42; Drexler, Leukemia (1996) 10(4), 588-99 and Ravandi et al., Clin Cancer Res. (2003) 9(2), 535-50).
Activation of these downstream signalling molecules by phosphorylation leads to the proliferative and pro-survival effects of FLT3 (Gilliland and Griffin (2002) and Levis and Small, Leukemia (2003) 17(9), 1738-52).
Somatic mutations of FLT3 involving internal tandem duplications in the juxtamembrane region of the receptor, or through point mutation of D835 in the activation loop have been demonstrated in approximately 30% of patients with acute myeloid leukaemia (AML), a cancer of the white blood cells caused through overproduction of immature myeloid white blood cells (Nakao et al., Leukemia (1996) 10(12), 1911-8; Thiede et al., Blood (2002) 99(12), 4326-35; Yamamoto et al., Blood (2001) 97(8), 2434-9; Abu-Duhier et al., Br. J. Haematol. (2000) 111(1), 190-5 and Abu-Duhier et al., Br. J. Haematol. (2001) 113(4), 983-8).
Other ligand independent activating mutations of FLT3 have recently been described, contributing to the leukaemic transformation in AML. Presence of such mutations at diagnosis has been linked to inferior prognosis in some patients (Jiang et al., Blood (2004) 104(6), 1855-8 and Kindler et al., Blood (2005) 105(1), 335-40).
FLT4
FLT4 is a receptor tyrosine kinase closely related in structure to the products of the VEGFR-1 and VEGFR-2 genes. FLT4 is activated by its ligand VEGF-C resulting in the promotion of angiogenesis and lymphangiogenesis (Alitalo and Carmeliet, Cancer Cell (2002) 1, 219-227; Plate, Nat. Med. (2001) 7, 151-152 and Skobe et al., Nat. Med., (2001) 7, 192-198).
FLT4 has been found to be expressed in a variety of human malignancies including lung adenocarcinoma (Li et al., Chin. Med. J. (2003) 116, 727-730), colorectal adenocarcinoma (Witte et al., Anticancer Res., (2002) 22, 1463-1466), prostate carcinoma (Kaushal et al., Clin. Cancer Res. (2005) 11, 584), head and neck carcinomas (Neuchrist et al., Head Neck (2003) 25, 464), leukaemia (Dias et al., Blood (2002) 99, 2179) and Kaposi's sarcoma (Weninger et al., Lab. Invest., (1999) 79, 243-251). Expression of FLT4 has also been shown to correlate with the different stages of cervical carcinogenesis (Van Trappen et al, J. Pathol., (2003) 201, 544-554).
Expression levels of VEGF-C and FLT4 were found to correlate with the stage and lymph node metastasis and survival of cancer patients with lung adenocarcinomas. The VEGF-C/FLT4 axis was shown to promote the migration and invasiveness of cancer cells (Kuo et al., 2006, Cancer Cell, 9, 209-223).
J. Lykkeberg et al., Acta Chemica Scandinavica, Series B: Organic Chemistry and Biochemistry (1975) B29(7), 793-5 describes the preparation of some 2,4-disubstituted imidazole-5-carboxamides by thermolysis of β-substituted α-(1-tetrazolyl)acrylamides. Amongst the compounds disclosed in the article are 2,5-diphenyl-1H-imidazole-4-carboxylic acid amide and 2-phenyl-5-thiophen-2-yl-1H-imidazole-4-carboxylic acid amide.
Ponomarev et al., Zhumal Fizicheskoi Khimii (1990) 64(10), 2723-9 (Chem Abs. 114:100938) describes the electronic absorption spectra of fused oxazole compounds. Amongst the compounds disclosed in the article is 2,5-diphenyl-oxazole-4-carboxylic acid amide.
Ozaki et al., Chem. Pharm. Bull. (1983) 31(12), 4417-24 discloses a series of 2-substituted oxazole compounds as blood platelet aggregation inhibitors. One of the compounds exemplified in the article is 2-phenyl-5-(3,4,5-trimethoxy-phenyl)-oxazole-4-carboxylic acid amide.
JP 63-10767 and JP 86-155456 (Yoshitomi) disclose diaryl imidazoles as analgesic and anti-inflammatory agents. The compound 2-(4-fluorophenyl)-5-(4-methoxyphenyl)-1H-imidazole-4-carboxylic acid amide is specifically disclosed.
WO 2006/095159 (AstraZeneca) discloses imidazolyl-anilino-pyrimidines as cell proliferation inhibitors.
WO 02/00649 (AstraZeneca) discloses substituted quinazolines as Aurora kinase inhibitors.
WO 2004/005283 (Vertex) discloses pyridyl and pyrimidinyl substituted oxazoles, thiazoles and imidazoles as protein kinase inhibitors.
WO 2007/043400 (Kissei) discloses aryl and heteroaryl pyrazole derivatives as xanthine oxidase inhibitors. The compound 2-(4-methylphenyl)-5-phenyl-oxazole-4-carboxylic acid amide is specifically disclosed as a chemical intermediate.
WO 2005/040139 (AB Science et al.) and WO 2007/131953 (AB Science) disclose 2-phenylamino-oxazoles as inhibitors of various tyrosine kinases.
WO 2008/024980 (Serenex Inc.) discloses pyrrole, thiophene, furan, imidazole, oxazole and thiazole derivatives that have Hsp90 inhibiting activity and which are useful for treating a range of diseases including cancer.